Kit for preparing Tc (III)-99m myocardial imaging agents that are effective in humans

ABSTRACT

A myocardial imaging agent for humans is a technetium(III) complex ligated in the planar positions by a tetradentate ligand such as (acac) 2  en and in the axial positions by an ether containing phosphine ligand such as tris(3-methoxy-1-propyl)-phosphine. The agent exhibits extremely rapid blood clearance after injection into a human and has a sufficiently high heart/liver and heart/lung ratios to provide effecitve myocardial images. A kit for preparing the above myocardial agent is disclosed.

This is a division of application Ser. No. 354,491, filed May 19, 1989,now U.S. Pat. No. 4,917,879.

BACKGROUND OF THE INVENTION

Several non-invasive methods of imaging body organs have been developedover the past decades. These procedures are based on the tendency of abody organ to concentrate some detectable chemical. Particularly usefulchemicals are those which emit gamma radiation. Subsequent scanning ofthe organ with a gamma ray camera provides an image of the organ fromwhich diagnostic information can be obtained. ^(99m) Tc (Tc-99m) hasbeen found to be particularly useful in this area because of itshalf-life and gamma ray emission.

Over the past several years different Tc-99m compounds have beendisclosed for use as positive myocardial imaging agents. These differentimaging agents, based on substantially different chemistries, haveexhibited varying levels of utility in different mammals. To effectivelyimage the heart the agent must localize in the heart and at the sametime rapidly clear from neighboring organs such as the lungs and inparticular the liver. Further, the imaging agent must not bind tightlyto the blood or else image quality will be poor. An imaging agent whichlocalizes in the heart and at the same time localizes in the liver doesnot provide a good image of the heart since the apex of the human heartis often obscured by the liver.

One recent patent which discloses Tc-99m myocardial imaging agents isDeutsch et al U.S. Pat. No. 4,795,626. This discloses a type ofmyocardial imaging agent which due to its ligand system is not reduciblein vivo. Thus, the disclosed Tc(III) complexes remain in this oxidationstate for imaging purposes. This has been found somewhat useful inmyocardial imaging.

Unfortunately, the prototypical agent of this class has relatively slowblood clearance and high liver uptake which gives rise to rather lowheart/liver ratio. This is reported in the Journal of Nuclear Medicine,28 1070 1000, 1987. The ligand system acac₂ en bonded to the four planarcoordinations sites of the technetium and PMe₃ (trimethylphosphine)bonded to the axial sites simply did not provide an efficaciousmyocardial imaging agent in humans.

One commercially acceptable product is Cardiolite sold by DuPont. Thisis an isonitrile Tc(I) complex. The isonitrile ligands contain alkylether groups. Also, Nuclear Medicine Communication, 10(4), Apr., 1989,p.245 reports myocardial imaging agents in which ^(99m) Tc is complexedto bidentate phosphorus ligands containing alkyl ether groups. Thisbrief abstract reports a heart/liver ratio of 0.75 which is lower thanwhat is required to obtain a good myocardial image.

SUMMARY OF THE INVENTION

The present invention is premised on the realization that Tc(III)myocardial imaging agents which are not reducible in vivo can be veryeffective myocardial imaging agents if the ligands contain one or moreether moietes in the ligand system.

More particularly, the present invention is premised on the realizationthat an effective myocardial imaging agent for humans can be prepared byligating a tetradentate ligand system to the 4 planar coordinationbonding sites of an octahedrally coordinated technetium center andbonding ligands containing ether moietes to the axial positions of thetechnetium center. Specifically, the present invention is premised onthe realization that a Tc(III) acac₂ en complex having alkyl ethersubstituted phosphine ligands at the axial positions provides acommercially viable heart imaging agent.

The present invention will be further appreciated in light of thefollowing detailed description and drawing in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing blood clearance from a human volunteer duringa stress evaluation of a myocardial imaging agent made according to thepresent invention.

FIG. 2 is a graph showing blood clearance from a human volunteer at restof a myocardial imaging agent made according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The technetium compounds which are useful as myocardial imaging agentsin humans are hexadentate technetium complexes which have an overallcationic charge. More specifically, the complexes will be technetiumcomplexes in the +3 oxidation state coordinatively bonded to six atomsas shown in Formula 1. ##STR1##

R' and R''' represent H, hydroxyl, C₁ -C₅ alkyl, C₁ -C₅ alkylsubstituted by hydroxyl, ether, amide, ketone, aldehyde or nitrilegroup.

R'' represents C_(1-C) ₄ alkylene, C_(1-C) ₄ alkenyl which may besubstituted with hydroxyl, ether, amide, ester, ketone, aldehyde ornitrile group.

The present technetium compound is bonded generally to three ligands,two axial ligands R₁ and R₂ as in Formula 1 and a tetradentate ligandhaving the following formula: ##STR2## The preferred tetradentate ligandis N,N'-ethylenebis(acetylacetone iminato) hereinafter referred to as(acac)₂ en wherein R'' represents methylene and all the R's representhydrogen and all the R'''s represent methyl. Also suitable tetradentateligands include N,N'-ethylenebis(tertbutylacetoacetate iminato)hereinafter referred to as (buac)₂ en, N,N'-ethylenebis(benzoylacetoneiminato) also referred to as (bzac)₂ en,N,N'-ethylenebis(3-bromoacetyacetone iminato) also referred to as(brac)₂ en and N,N'-methylethylenebis(acetylacetone iminato) alsoreferred to as (acac)₂ pn.

Ligands R₁ and R₂ also referred to as the axial ligands represent thesame or different ligands both falling within the following generalformula: ##STR3## wherein R₃ and R₄ represent a moiety having thefollowing general formulas 4 and 5: ##STR4## and wherein R₅ canrepresent the same moietes represented by R₃ and R₄ above or may inaddition represent --OCH₃, and C₁ -C₄ alkyl. Such a ligand can be madeaccording to the following example.

EXAMPLE 1

Standard procedures are used to convert 22.5 grams of3-methoxy-1-propylchloride (CH₃ OCH₂ CH₂ CH₂ Cl) and 4.9 g Mg metal tothe corresponding Grignard reagent in 110 mL tetrahydrofuran. To theGrignard reagent cooled in a dry ice acetone bath is slowly added 4.6 gof phosphorus trichloride in 40 mL of tetrahydrofuran. The reactionmixture is then allowed to warm to room temperature, and is subsequentlyheated at reflux for 30 min. This reaction mixture is then cooled to 10°C., and 70 mL of a saturated aqueous solution of ammonium chloride isadded. This hydrolyzed mixture is then filtered, and the aqueous layeris removed. The organic layer is dried over potassium carbonate andmagnesium sulfate, the tetrahydrofuran is removed by distillation, andthe desired phosphine product (P(CH₂ CH₂ CH₂₀ CH₃)₃ =TMPP) is recoveredby vacuum distillation (114-116° C. at 1.5 mm Hg). This phosphine isconverted to the hydrochloride adduct (P(CH₂ CH₂ CH₂ OCH₃ )₃+HCl=TMPP.HCl) with gaseous HCl (yield=5.8 g, 70%). 31-P NMR shows asingle peak at -29.844 ppm (vs. H3P04) for the free phosphine, and adoublet at 20.654, 16.406 ppm (vs. H₃ PO₄) for the hydrochloride adduct.FAB-MS (positive ion mode) shows a parent peak at 251 amu for thehydrochloride adduct.

The ligated technetium complex shown in Formula 1 is manufactured in atwo step process. A 99m-pertechnetate solution is obtained from a 99-Mogenerator. This method of obtaining ^(99m) Tc is well known to thoseskilled in the art and is disclosed for example in Deutsch et al U S.Pat. No. 4,489,054 incorporated herein by reference. This is alsodisclosed in Glavan et al U.S. Pat. No. 4,374,821 also incorporatedherein by reference. This pertechnetate can be diluted to the desiredradioactive concentration of 10-100 mCi/mL with normal saline.

The ^(99m) TcO₄ ⁻ (pertechnetare) in which Tc has an oxidation state of+7 is reduced to a technetium +5 complex having a formula ^(99m) Tc^(v)O(L)⁺. This is formed by heating ^(99m) TcO₄ ⁻ in the presence of thetetradentate ligand and a reducing agent such as stannous chloride orsodium borohydride. In the second step the ^(99m) Tc^(v) OL⁺ complex isfurther reduced by treating it with the axial ligand of Formula 3 atslightly elevated temperatures, i.e., heating the ^(99m) Tc(V) complexin the presence of the ligand. A chemical reducing agent such asborohydride salts, stannous ion salts or hyposulfite salts can also beadded.

The preparation of the Tc(V) complex is further described in Examples 2and 3 wherein the ligand is (acac)₂ en.

EXAMPLE 2 Preparation of ^(99m) Tc^(v) O(acac)₂ en⁺ in Ethanol

Pertechnetate is purified according to the method disclosed in U.S. Pat.No. 4,778,672. A C18 sep-pak cartridge was rinsed with 5 mL ethanol andthen 3 mL of 0.01 M tetrabutylammonium bromide in water. A desiredamount of ^(99m) TcO₄ ⁻ in saline was combined with 1 mL of 0.1 Mtetrabutylammonium bromide, mixed well, and passed slowly through theC18 Sep-pak. The Sep-pak was washed with 10 mL water, 10 mL air werepassed through, and the activity eluted with 1-2 mL ethanol.

A solution of 17 mg H₂ acac₂ en in 0.25 mL is combined with 1 mL of theabove tetrabutylammonium 99m-pertechnetate solution and the resultingsolution is deaereated for 15 minutes. Then 20 microliters of 1 M KOHand 10 microliters of a freshly prepared solution of 30 mg SnCl₂ in 20mL ethanol are added. The mixture is incubated at 90° C. for 15 minutes.

EXAMPLE 3 Preparation of ^(99m) TcO₂ (acac)₂ en⁺ in Water

17 mg of H₂ acac₂ en was dissolved in 0.1 mL of ethanol. Then 0.1 mL of^(99m) TcO₄ ⁻ and 0.9 mL of water was added and the mixture deareatedfor 15 minutes with scrubbed argon. 20 microliters of 1 M KOH and thereducing agent were added next. The best results were obtained with 2-20microliters of a solution of 0.1 mmole (19mg) stannous chloride in 20 mLH₂ O.

The mixture was heated for 15 minutes at 90° C. and cooled to roomtemperature. The reaction was monitored by HPLC on a PRP-1 column in 90%MeOH/0.01 M Na phosphate and 0.01 M Na heptanesulfonate (pH 7.0) at aflow rate of 1 mL/min. The Tc(V)0(acac₂ en)⁺ cation elutes at 4.0-4.2min. (As in all cases the positive charge of the cation is offset by abiologically acceptable anion such as chloride, as is well known.)

According to the present invention a myocardial imaging agent isprepared by reducing the ^(99m) Tc(V) complex as prepared in Examples 2and 3 to a ^(99m) Tc(III) complex. To accomplish this, the ^(99m) Tc(V)complex is combined with an ether substituted phosphine ligand such asP(CH₂ CH₂ CH₂ OCH₃)₃. A solution of the ligand is introduced, at ambientor elevated temperature. This acts to reduce the ^(99m) Tc(V) complex toa ^(99m) Tc(III) complex. The ^(99m) Tc(III) complex can then bepurified on cationic exchange resin or a reversed phase C₁₈ Sep-Pak. The99mTc(III) complex will have the structure of Formula 1.

This is further described in Examples 4 and 5.

EXAMPLE 4

0.3 mL of 0.1 M aqueous TMPP.HCl solution from Example 1 is added to the^(99m) Tc(V) preparation from Example 2 and the mixture incubated for 15minutes at 70° C.

The preparation is diluted with 20 mL deareated water (filtering may beneeded to remove precipitated ligand) and loaded on a C18 Sep-pak whichwas prewashed with 5 mL ETOH and 20 mL H₂ O. The cartridge is rinsedwith 20 mL H₂ O and then twice with 2 mL 80% ethanol-water. The compoundis eluted in 1-2 mL 80% ethanol-saline.

EXAMPLE 5

0.3 mL of a 0.1 M TMPP.HCl solution from Example 1 was added to the^(99m) Tc(V) preparation from Example 3 and incubated at 70° C. for 15minutes. This is then ready for use.

To demonstrate the effectiveness of the myocardial imaging agent formedusing the method of Example 4 about 13 millicuries of ^(99m) Tc activitywas injected into a human volunteer under stress (having exercised untilthe volunteer's heart rate was approximately 80% of maximum predicted bythe patient's age and physical condition). Blood samples were then takenimmediately after injection and for intervals up to 60 minutesthereafter. The blood clearance data are shown in FIG. 1. Likewise thesame test was conducted on a human volunteer at rest and the data areshown in FIG. 2.

This demonstrates extremely quick and effective blood clearance whichenables obtaining a clear useful myocardial image as soon as 5-10minutes after injection. In fact, due to the effective blood clearanceas well as the high heart/liver ratio very clear myocardial images,including computer assisted tomographic images, were obtained of thesevolunteers, making this a commercially acceptable positive myocardialimaging agent.

All the ^(99m) Tc(III) complexes described above are administeredintravenously as radiopharmaceuticals in a radioactive dose of from 0.01mCi/ml to 10 mCi/ml most preferably 2 mCi/ml-5mCi/ml. The administrationdose for humans is usually in the range 10-30 mCi.

Imaging of the heart can be carried out by scanning techniques afterwaiting an appropriate period of time to permit blood clearance of theradiopharmaceutical. For example, time dependent scintiscans of thechest region of a patient can be used. A computer interfaced 16 crystal,Ohio Nuclear Spectrometer can be used for these scans. The complexes ofthe present invention can also be used in single photon emissioncomputed tomography as described in Beyer et al, Diagnostic NuclearMedicine, Volume 1, No. 2, page 10 (summer of 1984).

The present invention is particularly suitable for use in a kitpreparation. The kit preparation would consist of two sterile, pyrogenfree vials, the first vial containing an effective ligand having thestructure shown in Formula 1 in combination with an effective reducingagent in this case the tin chloride. This would be a lyophilizedcomposition. The second vial would contain a protected salt of thephosphine ligand shown in Formula 3. Typically, this would be thephosphine salt bonded to HCl, H₂ SO₄, iron(II), copper(I) or zinc(II).The acid salts are preferred. The kit would be used by injecting thepurified 99m-pertechnetate obtained from a molybdenum generator into thefirst vial. This is heated as per Example 3. Saline is added to thesecond vial to dissolve the protected ligand. This saline solution isthen added to the first vial which is heated to effect conversion toTc(III). The contents of the first vial can be directly injected intothe patient without further purification.

The ^(99m) Tc(III) complexes of the present invention provide aradiopharmaceutical uniquely adapted for use in myocardial imaging ofhumans. These radiopharmaceuticals neither hang up in the blood systemnor the liver and yet bind to the heart for long periods of time (5h) toprovide useful positive human heart images.

Accordingly having described our invention, we claim:
 1. A kit forpreparing a technetium 99m myocardial imaging agent, said kitcomprising:a first and second vial, said first vial containing alyophilized pyrogen free sterile mixture of an effective reducing agentand a ligand having the following general formula: ##STR5## wherein R'and R''' represent H, hydroxyl, (C₁ -C₅ alkyl, C₁ -C₅ alkyl substitutedby hydroxyl, ether, amide, ketone, aldehyde or nitrile groups and R''represents C₁ -C₄ alkylene, C₁ -C₄ alkylene which may be substitutedwith hydroxyl, ether, amide, ketone, aldehyde or nitrile groups andwherein said second vial contains a lyophilized, pyrogen free, sterileprotected salt of a phosphine ligand, said phosphine ligand having thefollowing general formula: ##STR6## wherein R₄ and R₅ represent the sameor different group ##STR7## wherein X=1-4, Y=0-4 and Z=0-4 and whereinR₃ represents the same ligands represented by R₄ or R₅ or may represent:--OCH₃, --C₁ -C₅ alkyl.
 2. The kit claimed in claim 1 wherein said firstligand is H₂ acac₂ en.
 3. The kit claimed in claim 1 wherein saideffective reducing agent is tin chloride.
 4. The kit claimed in claim 1wherein said phosphine ligand comprises P(CH₂ CH₂ CH₂ OCH₃)₃.